Gut Online First, published on August 11, 2014 as 10.1136/gutjnl-2014-307329 Colon

ORIGINAL ARTICLE Gut: first published as 10.1136/gutjnl-2014-307329 on 11 August 2014. Downloaded from —regulated miR-133α is involved in proinflammatory signalling in colonic epithelial cells and in experimental colitis Ivy Ka Man Law,1 Kyriaki Bakirtzi,1 Christos Polytarchou,2 Angelos Oikonomopoulos,1 Daniel Hommes,1 Dimitrios Iliopoulos,2 Charalabos Pothoulakis1

▸ Additional material is ABSTRACT published online only. To view Objective Neurotensin (NT) mediates colonic Significance of this study please visit the journal online fl (http://dx.doi.org/10.1136/ in ammation through its 1 gutjnl-2014-307329). (NTR1). NT stimulates miR-133α expression in colonic α 1 fl epithelial cells. We investigated the role of miR-133 in What is already known on this subject? In ammatory Bowel Disease fl Center, Division of Digestive NT-associated colonic in ammation in vitro and in vivo. ▸ Neurotensin (NT) mediates colonic Diseases, David Geffen School Design miR-133α and aftiphilin (AFTPH) levels were inflammation through its high-affinity receptor of Medicine, UCLA, Los measured by quantitative PCR. Antisense (as)-miR-133α ( (NTR1)). Angeles, California, USA 2 was administrated intracolonicaly prior to induction of ▸ NT and NTR1 expressions are upregulated in Center for Systems fl Biomedicine, Division of 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)-induced in amed colon tissues in experimental colitis Digestive Diseases, David colitis and dextran sodium sulfate (DSS)-induced colitis. and ulcerative colitis (UC). Geffen School of Medicine, The effect of AFTPH was examined by silencing in vitro. ▸ NT modulates differential expression of 38 UCLA, Los Angeles, California, Results NT increased miR-133α levels in NCM-460 microRNAs in colonocytes, among them USA overexpressing NTR1 (NCM460-NTR1) and HCT-116 cells. miR-133α. κ Correspondence to NT-induced p38, ERK1/2, c-Jun, and NF- B activation, as ▸ MicroRNA-133α modulates signalling pathways Professor Charalabos well as IL-6, IL-8 and IL-1β messenger RNA (mRNA) related to proliferation, differentiation and Pothoulakis, Inflammatory expression in NCM-460-NTR1 cells were reduced in miR- hypertrophy in cancer cell lines and myocytes. Bowel Disease Center, Division 133α-silenced cells, while overexpression of miR-133α of Digestive Diseases, David reversed these effects. MiR-133α levels were increased in What are the new findings? Geffen School of Medicine, ▸ miR-133α modulates NT-induced MAP kinase, University of California at Los TNBS (2 day) and DSS (5 day) colitis, while NTR1 deficient α and NF-κB activation, as well as transcription Angeles, 675 Charles E Young DSS-exposedmicehadreducedmiR-133 levels, compared http://gut.bmj.com/ Dr, South MRL building 1240, to wild-type colitic mice. Intracolonic as-miR-133α attenuated of proinflammatory cytokines in vitro. Los Angeles, CA 90095, USA; several parameters of colitis as well expression of ▸ Reducing the levels of miR-133α in human [email protected] proinflammatory mediators in the colonic mucosa. In silico colonocytes and mouse colon attenuates fl Received 26 March 2014 search coupled with qPCR identified AFTPH as a proin ammatory signalling and development of Revised 2 July 2014 downstream target of miR-133α, while NT decreased AFTPH experimental colitis in vitro and in vivo. Accepted 22 July 2014 expression in NCM-460-NTR1 colonocytes. Gene silencing of ▸ Aftiphilin (AFTPH) is a novel downstream target

AFTPHenhancedNT-inducedproinflammatory responses and of NT-induced miR-133α expression in human on September 29, 2021 by guest. Protected copyright. AFTPH levels were downregulated in experimental colitis. colonic epithelial cells involved in Levels of miR-133α were significantly upregulated, while proinflammatory signalling pathways. AFTPH levels were downregulated in colonic biopsies of ▸ Colon tissues from patients with UC and mice patients with ulcerative colitis compared to controls. with colitis have higher miR-133α but lower Conclusions NT-associated colitis and inflammatory AFTPH levels when compared with controls. α signalling are regulated by miR-133 -AFTPH interactions. How might it impact on clinical practice in Targeting of miR-133α or AFTPH may represent a novel fl the foreseeable future? therapeutic approach in in ammatory bowel disease. ▸ Inhibiting miR-133α and restoring AFTPH expression in inflammatory bowel disease (IBD) colon may represent promising approaches for the treatment of IBD. INTRODUCTION ▸ The reverse association of the levels of Neurotensin (NT) is a expressed in miR-133α and AFTPH together with increased 12 the and the intestine, expression of NTR1 in UC may represent novel while its high-affinity receptor, NT receptor 1 biomarkers for UC. (NTR1)3 is expressed in neurones,4 colonic epithe- – lial,257 immune cells,89as well as different colon cancer cell lines.10 In the colon, NT/NTR1 signal- To cite: Law IKM, ling promotes inflammation in acute colitis animal ulcerative colitis (UC).6 Moreover, NTR1 defi- Bakirtzi K, Polytarchou C, 25 et al. Gut Published Online models. Colonic NT and NTR1 expression ciency in mice is associated with reduced weight 513 First: [please include Day increases during enterotoxin-mediated intestinal loss and mortality in experimental colitis. Month Year] doi:10.1136/ inflammation,2 dextran sodium sulfate (DSS) MicroRNAs (miRs) are short (19–25 nucleo- gutjnl-2014-307329 colitis,61112and in the colon of patients with tides), single-stranded RNA molecules, acting as

Copyright Article author (or their employer)Law IKM, et al .2014.Gut 2014; Produced0:1–10. doi:10.1136/gutjnl-2014-307329 by BMJ Publishing Group Ltd (& BSG) under licence.1 Colon negative transcriptional regulators. They bind to the 30 untrans- phosphoprotein detection, and media were collected 6 h after Gut: first published as 10.1136/gutjnl-2014-307329 on 11 August 2014. Downloaded from lated regions (UTR) of transcripts14 and lead to messenger RNA stimulation for cytokine neasurements. Custom designed (mRNA) degradation, or inhibition of translation into .15 Bio-Plex Pro Cell Signalling assay panel (Bio-Rad, Hercules, Recently, several studies identified the differential miR expres- Californai, USA) and Bio-Plex Pro Human Cytokine 27-plex – sion in colonic mucosa in patients with UC,16 19 Crohn’s Assay (Bio-Rad) were used for phosphoprotein activation and Disease (CD)18 20 and experimental colitis.17 However, the role cytokine production (except IL-8), respectively, according to of individual miRs in inflammatory bowel disease (IBD) patho- manufacturer’s instructions. physiology is still under investigation. We have recently shown that NT/NTR1 coupling in human Animal models and establishment of experimental colonic epithelial NCM460 overexpressing NTR1 colitis models (NCM460-NTR1) cells induces differential expression of Neurotensin receptor 1 knockout mouse model 10 miRs, including miR-133α. Dysregulation of miR-133α and Ntsr1tmDgen (hereafter called NTR1KO) were purchased from its target has been correlated with colorectal cancer devel- Jackson Laboratories and bred in our facility. Animals were 21–28 29 30 21 31 opment through regulating ERK and Akt activa- obtained as fifth generation backcross of 129 onto C57BL6/J. tion, pathways also targeted by NT/NTR1 signalling in human We performed one additional backcross before intercrossing 71032 colonic epithelial cells. Based on these considerations, we animals as heterozygous NTR1KO crosses to generate littermate examined the role of NT-miR-133α interactions in colonic controls. inflammation in vitro and in two experimental colitis models induced by trinitrobenzene sulfonic acid (TNBS) and DSS in TNBS-induced colitis vivo. We also performed studies to identify the target of C57BL6/J wild-type (WT) mice were purchased from Jackson α NT-NTR1-modulated miR-133 that mediates these effects. Laboratories. TNBS was dissolved in 30% ethanol (5 mg/kg) Our results point to aftiphilin (AFTPH), as a novel downstream was intracolonicaly administered (50 μL) to mice as previously α target for miR-133 , and present evidence for the association of described.533Briefly, TNBS was slowly infused via a 1 mL α miR-133 and its downstream target, AFTPH, in the patho- syringe (Becton Dickinson, Laguna Hills, California, USA) fitted physiology of colitis. with a polyethylene cannula (Intramedic PE-20 tubing; Becton Dickinson). Colon tissues were collected 48 h after TNBS MATERIALS AND METHODS administration. Cell culture and reagents NCM460-NTR1 cells were generated from human colonic epi- DSS-induced colitis thelial NCM460 cells (INCELL, San Antonio, Texas, USA) NTR1KO and its WT counterparts, or C57BL6/J WT animals, transduced with lentivirus particles as previously described10 were treated with 5% DSS in drinking water as previously and maintained in M3:D culture media (INCELL) supplemen- described,13 34 and colon tissues were collected 5 days after the ted with 10% fetal bovine serum (FBS, Life Technologies, start of the treatment. Grand Island, New York, USA). Human embryonic kidney fibro- http://gut.bmj.com/ In vivo NT infusion. NT was dissolved in PBS supplemented blasts, HEK293, and colonic cancer HCT-116 cells were main- with 1% bovine serum albumin (BSA)35 and was administered tained in Eagle’s Minimum Essential Medium (MEM, Life intracolonicaly (300 μg/kg) to C57BL6/J WT mice twice per day Technologies) and McCoy5a (ATCC, Manassas, Virginia, USA), for 4 days. Control mice were infused with 1% BSA in PBS. respectively, and supplemented with 10% FBS. Lipofectamine Colon tissues were collected 5 days after NT administration. 2000, lipofectamine RNAimax and OptiMEM were from Life Technologies. NT was from Bachem Americas (Torrance,

α on September 29, 2021 by guest. Protected copyright. California, USA). TNBS and SR48962 were from Sigma Aldrich In vivo knockdown of miR-133 α (St Louis, Missouri, USA), and DSS was from MP Biomedicals Endogenous expression of miR-133 in colon tissues of (Santa Ana, California, USA). Goat anti-AFTPH (sc-167055), C57BL6/J WT mice was silenced by intracolonic administration mouse antivillin and rabbit anti-β tubulin were from Santa Cruz of miRCURY LNA Inhibitor probe, in vivo against mmu-miR-133a μ fl Biotechnology (Santa Cruz, California, USA). (20 g/mouse, Exiqon). Brie y, the appropriate amount of oligonucleotides against mmu-miR-133a and its μ Transfection experiments respective control were resuspended in 100 L Opti-MEM with μ Small interfering RNA (siRNA) was purchased from Santa Cruz 2 L lipofectamine 2000 and administered intracolonicaly 24 h Biotechnology (Santa Cruz). NCM460-NTR1cells were trans- and 72 h prior to TNBS or DSS treatment. fected with siRNA against AFTPH (si-AFTPH) using Lipofectamine RNAiMAX. All miRs were purchased from Life MicroRNA in situ hybridisation Technologies. For miR-133α silencing or overexpression, cells Colon tissues (3 cm from distal end) were obtained from WT were transfected with antisense-miR-133α (as-miR-133α)or mice 2 day after TNBS treatment. The tissues were immediately miR-133α precursor (miR-133α), respectively, using fixed with 4% paraformaldehyde, paraffin-embedded and sec- 0 0 Lipofectamine RNAiMAX. Cells transfected with siRNA-A tioned (5 μm) for histological study. 5 -DIG and 3 -DIG labelled (si-control), antisense-control miR (as-miR-control) or detection probes specific for mouse miR-133α (Cat. No. miR-negative control precursor (miR-control) served as controls. 39270-15) and miRCURY LNA microRNA ISH optimisation Kit (FFPE) were purchased from Exiqon, and the experiments NT/NTR1-mediated phosphoprotein activation were performed according to the manufacturer’s instructions. and cytokine production NCM460-NTR1 cells were transfected with as-miR-133α, RNA expression studies in patient samples miR-133α and si-AFTPH and their respective controls. Cells Total RNAs from colon tissues obtained from patients with UC were incubated in serum-free media overnight and stimulated (n=12), CD (n=8) and normal subjects (n=9) were purchased with NT (100 nM). Cell lysates were collected for from Origene (Rockville, Maryland, USA). Conversion of cDNA

2 Law IKM, et al. Gut 2014;0:1–10. doi:10.1136/gutjnl-2014-307329 Colon of RNA samples was performed as described above, and levels Gut: first published as 10.1136/gutjnl-2014-307329 on 11 August 2014. Downloaded from of miR-133α and AFTPH were determined by qPCR analysis.

Immunohistochemistry Frozen tissue sections (5 μm) obtained from chronic active patients with UC (n=3) and their normal control (n=3) were purchased from Origene. The tissues were fixed with 4% paraf- ormaldehyde and blocked with 0.3% Triton X-100 in PBS (PBS-Triton) supplemented with 5% normal bovine serum. Appropriate antibodies were incubated with the tissue sections overnight at 4°C, washed with PBS-Triton and incubated with appropriate secondary antibodies (Santa Cruz Biotechnology). The tissues were imaged using Axio Imager.Z1 microscope Figure 1 Levels of miR-133α were upregulated after neurotensin (NT) (Carl Zeiss). exposure in human colonic epithelial cells. (A) Expression of miR-133α Please refer to online supplementary methods for site-directed in human colonic epithelial NCM460 cells after NT exposure was mutagenesis, luciferase assay, NF-κB p65 translocation, measure- analysed by qPCR analysis. **p<0.01, compared to vehicle control. (B) miR-133α levels in human colon cancer HCT-116 cells after NT ment of interleukin-8 (IL-8) production, immunoblot analysis, fi exposure were analysed by qPCR analysis. **p<0.01, compared to mRNA and miR expression analysis and microRNA pro ling in vehicle control. blood samples.

Statistical analysis All in vitro results derived from at least three sets of experi- NT-induced NF-κB p65 phosphorylation was blocked in ments, expressed as means±SD and analysed with Student t as-miR-133α-transfected cells (figure 2C), while NT-induced tests. Results from animal experiments and studies in human IL-6 and IL-8 mRNA (figure 2D, E, p<0.05) and IL-8 secretion tissues were analysed with Student t tests and expressed as were also significantly attenuated upon miR-133α knock-down means±SEM. In all statistical comparisons, p<0.05 was used to (figure 2F, p<0.05). Analysis of the cell culture media showed indicate significant differences. that NT stimulation increased the production of the proinflam- matory IL-1β, but reduced the production of the anti- RESULTS inflammatory IL-1ra (figure 2G, p<0.05). Transfection of Increased miR-133α levels in human colonic epithelial cells as-miR-133α significantly reduced NT-induced IL-1β production, after NT exposure while prevented the decrease in IL-1ra production (figure 2G). – NT exerts its effect on colonic epithelial cells257 through its Together, these results suggest an important role for miR-133α in high-affinity receptor NTR1.3 NTR1 overexpression is observed the induction of proinflammatory signalling pathways and cyto- 10 36 in colon cancer cell lines, colon tissues from mice with kine production in response to NTR1 activation in human http://gut.bmj.com/ experimental colitis,61112intestinal tumors37 and in tissue colonocytes. biopsies from patients with UC.6 In a microarray analysis, we We further verified the proinflammatory role of NT and previously showed that NT/NTR1 coupling in human colonic miR-133α in NCM460 cells transfected with NTR1 and epithelial NCM460-NTR1 increased miR-133α expression.10 In miR-133α. Overexpression of miR-133α further increased our current study, we first verified this result by examining the NT-induced ERK1/2, c-Jun (figure 3A, p<0.05) and NF-κB p65 levels of miR-133α in human colonic epithelial (figure 3B, p<0.05) activation. Additionally, overexpression of NCM460-NTR1 cells and in colon cancer adenocarcinoma miR-133α alone, in the absence of NT stimulation, promoted on September 29, 2021 by guest. Protected copyright. HCT-116 cells by qPCR. Consistent with our previous find- nuclear translocation of NF-κB p65 (figure 3C, p<0.05) when ings,10 levels of miR-133α were upregulated after NT exposure compared to the cells expressing control miR. Furthermore, by 2.6±0.43-fold (p<0.01, figure 1A) in NCM460-NTR1 cells, IL-8, IL-1β and TNF-α mRNA was increased in cells overexpres- while in HCT-116 cells, levels of miR-133α were increased by sing miR-133α when compared to their controls (figure 3D, 1.5±0.07-fold (p<0.01, figure 1B) after addition of NT. p<0.05). For cytokine production, IL-8 secretion was increased Therefore, NT increases miR-133α expression in two different upon miR-133α overexpression (figure 3E, p<0.05). Cells over- human colonic epithelial cell lines. expressing miR-133α had also significantly reduced IL-1ra pro- duction (p<0.05), while IL-1β production was increased NT induces proinflammatory signalling in human colonic slightly, but not significantly (figure 3F). Together, these results epithelial cells via miR-133α expression strongly suggest an important role for miR-133α in proinflam- NT coupling of NTR1 stimulates inflammatory responses matory signalling in human colonocytes. through MAPK38 39-dependent and NF-κB74041-dependent pathways. Here, we examined whether miR-133α is involved in NT-related proinflammatory signalling and cytokine expression MiR-133α overexpression in vivo is NT/NTR1-dependent in NCM460-NTR1 cells with an antisense approach using Since, as shown above, NT regulates miR-133α in colonocytes as-miR-133α. First, we showed that transfection of as-miR-133α in vitro, we next examined whether this effect can be recapitu- blocked NT-induced miR-133α overexpression in lated in vivo. To do this, C57/BL6J WT (WT) mice received NCM460-NTR1 cells (figure 2A), compared to those trans- intracolonicaly NT (300 μg/kg).35 After 4 days, mice were eutha- fected with control as-miR, validating this approach. Moreover, nased and colonic tissues were processed for RNA purification. NT stimulation activated p38 and ERK1/2 signalling only in qPCR analysis showed increased colonic miR-133α levels (by cells transfected with control as-miR, but not as-miR-133α, 1.3±0.04-fold, figure 4A, p<0.05) in mice administered with while NT-induced c-Jun activation was significantly attenuated NT, compared to intracolonic administration of vehicle. Because in as-miR-133α-transfected cells (figure 2B, p<0.05). Moreover, NT is an important mediator of acute colonic inflammation,213

Law IKM, et al. Gut 2014;0:1–10. doi:10.1136/gutjnl-2014-307329 3 Colon

Figure 2 Downregulation of Gut: first published as 10.1136/gutjnl-2014-307329 on 11 August 2014. Downloaded from miR-133α attenuated neurotensin (NT)-associated proinflammatory signalling in NCM460-neurotensin receptor 1 (NTR1) cells. (A) Levels of miR-133α in NCM460-NTR1 cells upon NT stimulation after transfection of as-miR-133α and its control were analysed by qPCR analysis. *p<0.05, compared to cells transfected with as-miR-control. (B) Levels of MAP kinase phosphorylation, including p38, ERK1/2 and c-Jun and (C) NF-κB phosphorylation in NCM460-NTR1 cells transfected with as-miR-133α and its control were analysed as stated in Methods. *p<0.05, compared to cells transfected with as-miR-control; ##p<0.01, compared to cells treated with NT. (D) IL-6 and (E) IL-8 levels in NCM460-NTR1 cells transfected with as-miR-133α and its control was analysed by qPCR analysis. **p<0.01, compared to cells transfected with # as-miR-control and p<0.05, compared with NT-treated cells. (F) IL-8 production in NCM460-NTR1 cells transfected with as-miR-133α and its control was measured using ELISA. **p<0.01, compared to cells treated with vehicle control; #p<0.05, compared with NT-treated cells. (G) IL-1β and IL-1ra production in NCM460-NTR1 cells transfected with as-miR-133α and its control was measured as stated in Methods.

*p<0.05, compared to cells treated http://gut.bmj.com/ with vehicle control. we also examined colonic levels of expression of miR-133α in miR-133α expression during acute colitis involves NT/NTR1 two colitis models. Acute colitis was induced in C57BL6/J WT interactions. mice by intracolonic administration of 5 mg/kg TNBS, or by addition of 5% DSS in their drinking water. qPCR analysis Intracolonic silencing of miR-133α attenuates the on September 29, 2021 by guest. Protected copyright. showed increased miR-133α levels (by 3.2±1.01-fold, figure 4B, development of acute colitis p<0.05) 2 days after TNBS administration, but decreased by We next determined the role of miR-133α in the development 63% at day 7 (figure 4B, p<0.05). Mice receiving DSS showed of experimental colitis. C57BL6/J WT mice were administered a 3.6±0.61-fold increase in miR-133α levels (figure 4B, intracolonicaly two doses of as-miR-133α or its control 24 h p<0.001) at the end of the DSS treatment, compared to and 72 h prior to intracolonic administration of TNBS (5 mg/kg) controls. or vehicle. Colonic tissues were harvested 48 h To study the mucosal distribution of miR-133α in colon post-TNBS-treatment for and histological ana- tissue during colitis, in situ hybridisation against miR-133α in lysis. Histological examination on colon tissues collected from colon tissue sections obtained from TNBS-treated WT mice and mice without TNBS-treatment did not show any differences their control counterparts was performed as described in (figure 5A). As expected, TNBS administration induced acute Methods. Our result show that while miR-133α expression was colonic inflammation in mice administered with as-miR-control, higher in inflamed colons, the majority of miR-133α expression while administration of as-miR-133α prior to TNBS treatment was localised in epithelial cells (figure 4C, arrows) and less in attenuated colitis development (figure 5A). Additionally, quantita- other cells of the lamia propria (figure 4C, arrowheads). tive PCR analysis showed that intracolonic administration of The direct relationship of endogenous NT/NTR1 signalling as-miR-133α significantly reduced endogenous colonic expression on miR-133α expression in colon tissues during colitis develop- of miR-133α (p<0.05, figure 5B). Mice treated with ment was further investigated in NTR1-deficient NTR1KO as-miR-133α and TNBS showed a significant reduction in colon mice. NTR1KO mice and their WT counterparts were allowed weight after normalisation against colon length (figure 5C, access to 5% DSS in their drinking water, and after 5 days the p<0.05), neutrophil infiltration, and an improvement in mucosal mice were euthanased and miR-133α levels were measured. As integrity which resulted in a significantly improved histological shown in figure 4D, colonic miR-133α levels were increased in score (figure 5D, p<0.05). Proinflammatory cytokine production WT mice after DSS treatment (2.4±0.32-fold, p<0.01), but was generally increased in colon tissues treated with TNBS. decreased in NTR1KO mice (p<0.05). Thus, increased Pretreatment with as-miR-133α prior to TNBS administration

4 Law IKM, et al. Gut 2014;0:1–10. doi:10.1136/gutjnl-2014-307329 Colon

Figure 3 Overexpression of Gut: first published as 10.1136/gutjnl-2014-307329 on 11 August 2014. Downloaded from miR-133α promoted proinflammatory signaling in NCM460-neurotensin receptor 1 (NTR1) cells. (A) Levels of MAP kinase phosphorylation, including ERK1/2 and c-Jun and (B) NF-κB p65 phosphorylation in NCM460-NTR1 cells transfected with miR-133α and its control was analysed as stated in Methods. *p<0.05, compared to cells transfected with control miRNA precursor. (C) Translocation of NF-κB p65 in NCM460-NTR1 cells transfected with miR-133α and its control was analysed by ELISA. *p<0.05, compared to cells transfected with control miRNA precursor. (D) Expression of IL-8, IL-1β and TNF-α in NCM460-NTR1 cells transfected with miR-133α and its control was analysed by qPCR analysis. *p<0.05, compared to cells transfected with control miRNA precursor. (E) IL-8 production in NCM460-NTR1 cells transfected with miR-133α and its control was measured using IL-8 ELISA. *p<0.05, compared to cells transfected with control miRNA precursor. (F) IL-1β and IL-1ra production in NCM460-NTR1 cells transfected with miR-133α and its control was measured as stated in Methods. *p<0.05, compared to cells treated with vehicle control; #p<0.05, compared to cells transfected with miR control precursor. http://gut.bmj.com/ blocked increased expression of the neutrophil product lipocalin The NTR1-miR-133α-AFTPH interaction was first validated 2 (lcn2), and TNF-α, a major proinflammatory cytokine in in NCM460-NTR1 cells transfected with as-miR-133α and colitis. The production of cxcl1, an important neutrophil chemo- exposed to NT. NT exposure downregulated AFTPH mRNA attractant (figure 5E, p<0.05) was significantly reduced in mice levels, as shown in quantitative PCR analysis (figure 6B, administered with as- miR-133α, when compared to control p<0.05) and AFTPH 30UTR-regulated luciferase activity assay as-miR-administered counterparts (figure 5E). We also investi- (figure 6C, p<0.05) when compared with control cells. To gated the effect of as-miR-133α treatment in the DSS-induced confirm the direct interaction between miR-133α and AFTPH, on September 29, 2021 by guest. Protected copyright. colitis model. Administration of as-miR-133α and its control was we deleted the miR-133α binding sequence on the AFTPH 30 performed at 24 h and 72 h before DSS treatment. Mice adminis- UTR and showed that NT-induced downregulation of AFTPH tered with as-miR-133α and DSS showed reduced weight loss 30 UTR luciferase activity was not observed in the absence of 5 days after DSS treatment (see online supplementary figure S1A, the miR-133α binding site (figure 6D). Overexpression of p<0.05) while levels of TNF-α and IL-1β were increased 5 days miR-133α in HEK293 cells significantly reduced AFTPH-30 after DSS-induced colitis and significantly reduced in mice admi- UTR-associated luciferase activity in the absence of NT stimula- nistered with as-miR-133α (see online supplementary figure S1B, tion (figure 6E, p<0.05). Moreover, treatment with the C, p<0.05). In conclusion, these results suggested that miR-133α NTR1-specific antagonist of SR48962 prevented the reduction is involved in the pathophysiology of colitis. in AFTPH protein expression induced by NT (figure 6F). The above results strongly indicate that AFTPH is a downstream target of miR-133α in colonocytes after NT exposure, suggest- MiR-133α directly regulates AFTPH expression through ing that NT/NTR1 signalling suppresses AFTPH expression binding its 30 UTR in colonic epithelial cells through miR-133α overexpression. MiRs act as gene-silencers by inhibiting the expression of To confirm our in vitro findings in the in vivo setting, we mRNA transcripts through binding to the 30 UTRs of target compared expression of AFTPH mRNA in colon tissues from genes.14 To identify candidate genes involved in the control, NT-exposed and TNBS-exposed (2d) mouse colon. Our NTR1-miR-133α response, we searched for genes with possible results show significantly decreased colonic AFTPH mRNA miR-133α binding sites in their 30 UTRs. In silico search using expression in NT-administered (figure 6G, p<0.05) and three online databases (TargetScanHuman (http://www. TNBS-injected mice (figure 6H, p<0.05) compared to targetscan.org); miRBase (http://www.mirbase.org) and PicTar vehicle-exposed control colon, consistent with the increased (http://pictar.mdc-berlin.de)), identified aftiphilin (AFTPH) with miR-133α levels observed in both models (figures 4A and 5F). a miR-133α binding site at its 30 UTR highly conserved across In NTR1KO mice exposed to DSS, only a small (by 1.28 species (figure 6A). ±0.17-fold, n=5 per group) but not significant increase in

Law IKM, et al. Gut 2014;0:1–10. doi:10.1136/gutjnl-2014-307329 5 Colon

Figure 4 Levels of miR-133α were Gut: first published as 10.1136/gutjnl-2014-307329 on 11 August 2014. Downloaded from upregulated during experimental colitis in vivo. (A) miR-133α levels in colon tissues from C57BL6/J mice intracolonically administered with NT (300 μg/kg) was analysed by qPCR analysis. *p<0.05, compared to mice receiving vehicle control. (B) Expression of miR-133α in colon tissues from C57BL6/J mice collected 2 and 7 days after 5 mg/kg trinitrobenzene sulfonic acid (TNBS) administration and 5 days after 5% dextran sodium sulfate (DSS) feeding was analysed by qPCR analysis. *p<0.05, compared to treatment control. (C) Representative images of in situ hybridisation of miR-133α of colon tissues from TNBS-treated C57BL6/J mice and their control counterparts (arrows, epithelial cells; arrow heads, lamina propria cells infiltration). Scale: 50 μm. (D) Expression of miR-133α in colon tissues from neurotensin receptor 1 (NTR1) KO and its control counterparts collected 5 days after 5% DSS feeding was analysed by qPCR analysis. *p<0.05, compared to wild-type mice with same treatment.

AFTPH levels were observed. Along these lines, silencing of colonic mucosa when compared with normal colon tissues miR-133α by intracolonic administration of as-miR-133α (figure 8B), with AFTPH expressed primarily in colonic epithe- increased AFTPH levels in normal and inflamed colon tissues, lial cells, and colocalised with villin, an epithelial cell marker when compared to their control counterparts (figure 6H, (figure 8B). The above results suggested that as in mouse colitis, http://gut.bmj.com/ p<0.05). miR-133α and AFTPH expression are inversely dysregulated in patients with UC. AFTPH gene silencing promotes colonic proinflammatory responses in human colonic epithelial cells DISCUSSION To examine the functionality of AFTPH in NT-mediated proin- Coupling of NT to its high-affinity receptor NTR1 triggers

fl on September 29, 2021 by guest. Protected copyright. ammatory signalling, we silenced expression of AFTPH in MAPK38 39 and NF-κB74041signalling and promotes intes- NCM460-NTR1 cells with si-RNA transfection prior to NT tinal inflammation.2513We have previously shown that NT stimulation. Our results show that AFTPH gene silencing alters expression of several microRNAs, including miR-133α κ fi enhanced NT-induced c-Jun and NF- B activation ( gure 7A, B, in human colonic epithelial cells, and presented evidence that fi α p<0.05), consistent with our ndings for a role of miR-133 in microRNAs represent an important functional component of NT-mediated signalling activation. Additionally, gene silencing the effects of NT in the intestine.10 In this current study, we β fi of AFTPH increased basal IL-1 production ( gure 7C, examined the role of miR-133α in the NT-associated intes- p<0.05), while ameliorated NT-mediated IL-1ra reduction in tinal inflammatory circuit in vitro and in vivo. Our results fi human colonic epithelial cells ( gure 7C), in line with our demonstrate that expression of miR-133α is increased follow- α fi results from miR-133 overexpression ( gure 3F). ing NT stimulation of colonocytes in vitro and normal mouse colon in vivo. We also present strong evidence that miR-133α Levels of miR-133α and AFTPH are dysregulated in colon is involved in the inflammatory cascade activated by NT/ of patients with UC NTR1 interactions in human colonocytes, and that silencing To confirm our mouse findings, we compared levels of of endogenous miR-133α in the colon diminishes acute miR-133α and AFTPH in colonic tissue samples from UC and experimental colitis in two different mouse models. Last, our normal colon tissues. Levels of miR-133α were significantly results point to AFTPH as a novel downstream target of increased by 2.6±1.82-fold (p=0.048, figure 8A) in patients miR-133α that mediates intestinal proinflammatory signalling with UC with active disease, while AFTPH mRNA levels were and cytokine transcription following NT-miR-133α interac- decreased by 0.72±0.17-fold (p=0.0073, figure 8A) in the tions. Thus, the NT-regulated miR-133α and its target same patients. By contrast, levels of miR-133α obtained from AFTPH may represent new targets for colonic inflammatory patients with CD did not show significant difference compared responses in vivo. with normal controls (n=8 per group, data not shown). Our present study focused on understanding the role of Additionally, immunohistochemistry of colon tissues from miR-133α in NT-related colonic inflammation. NT/NTR1 sig- patients with UC showed reduced AFTPH expression in the nalling promotes acute colitis,25while NTR1 deficiency

6 Law IKM, et al. Gut 2014;0:1–10. doi:10.1136/gutjnl-2014-307329 Colon

Figure 5 Intracolonic administration Gut: first published as 10.1136/gutjnl-2014-307329 on 11 August 2014. Downloaded from of as-miR-133α attenuated trinitrobenzene sulfonic acid (TNBS)-induced colitis development in wild-type mice. (A) Representative images from H&E staining of colon tissues from C57BL6/J mice intracolonically administered with as-miR-133α and its control with or without TNBS treatment. Scale: 50 μm. (B) Levels of miR-133α in colon tissues from C57BL6/J mice intracolonically administered with as-miR-133α and its control were analysed using qPCR analysis. *p<0.05, compared to mice administered with control as-miRNA. (C) Colon weight from C57BL6/J mice intracolonically administered with as-miR-133α and its control with or without TNBS treatment were measured and normalised by colon length. *p<0.05, compared to mice administered with control as-miRNA. (D) Total histological score, neutrophil infiltration and mucosal integrity of colon tissues from C57BL6/J mice intracolonically administered with as-miR-133α and its control with TNBS treatment were scored. *p<0.05, **p<0.01, ***p<0.005 compared to mice administered with control as-miRNA. (E) Levels of lcn2, TNF-α and cxcl1 in colon tissues from C57BL6/J mice intracolonically administered with as-miR-133α and its control with or without TNBS treatment were analysed using http://gut.bmj.com/ quantitative PCR analysis. *p<0.05, compared to mice administered with control as-miRNA. on September 29, 2021 by guest. Protected copyright. reduces the severity of experimental colitis.513Additionally, proinflammatory signalling pathways by modulating miR-133α NTandNTR1areoverexpressedincolontissuesfrom levels in human colonic epithelial cells. patients with UC,6 and as shown in our current study, Our results showed that miR-133α levels were significantly NTR1KO mice have reduced miR-133α colonic levels com- increased in the colonic mucosa of mice with acute pared to wild-type mice. On the other hand, miR-133α and TNBS-induced and DSS-induced colitis, as well as colon tissues its downstream target expression have been associated with of patients with UC with active disease, compared to controls. – different disease states, such as colorectal cancer,21 28 MiR-133α was primarily expressed in colonic epithelial cells as – myocyte development, and hypertrophy,42 44 and fibrosis in shown by in situ hybridisation in mouse colon, with increased the cardiac muscle and liver.45 46 However, its role in colonic expression of this miR during TNBS-induced colitis. However, inflammation has never been studied. Several studies have miR-133α was not detectable in sera from patients with UC and shown that miR-133α modulates ERK29 30 and PI3 K/Akt21 31 their normal counterparts (data not shown). To study the role of signalling pathways, or directly targets molecules involved in endogenous miR-133α in colitis, we introduced as-miR-133α these pathways.22 Consistent with the above findings, we have intracolonicaly in the two experimental colitis models to avoid shown that miR-133α modulates ERK1/2 and p38 activation perturbation of normal cellular functions of non-target tissues. upon NT stimulation. We also demonstrate for the first time Our results showed that mice receiving intracolonic that miR-133α directly regulates NF-κB activation and NT as-miR-133α had reduced endogenous miR-133α levels, exposure, together with miR-133α overexpression, in cells improved mucosa histology and reduced proinflammatory cyto- increased the production of proinflammatory IL-1β and sup- kine expression in colon tissues of mice with colitis. pressed that of the anti-inflammatory IL-1ra. IL-1ra antago- Intracolonic delivery of antisense oligonucleotides is largely con- nises IL-1β in colitis in vivo.47 48 Interestingly, a reduced fined to colonic epithelial cells in vivo,50 our results suggest that IL-1ra/IL-1β ratio is considered as an important factor for the miR-133α levels in colonic epithelial cells can influence the severity of inflammation in patients with IBD.49 Taken pathophysiology of colitis. However, since expression of together, our results suggest that NT/NTR1 coupling triggers miR-133α has also been identified in circulating monocytes,51

Law IKM, et al. Gut 2014;0:1–10. doi:10.1136/gutjnl-2014-307329 7 Colon Gut: first published as 10.1136/gutjnl-2014-307329 on 11 August 2014. Downloaded from

Figure 7 Gene silencing of aftiphilin (AFTPH)-promoted neurotensin (NT)-associated proinflammatory response in vitro. Levels of (A) c-Jun and (B) NF-κB phosphorylation in NCM460-neurotensin receptor 1 (NTR1) cells transfected with si-AFTPH and its control was measured as stated in Methods. *p<0.05, compared to cells transfected with control siRNA; #p<0.05, compared to NT-treated cells. (C) IL-1β and IL-1ra production of NCM460-NTR1 cells transfected with si-AFTPH and its control in the presence or absence of NT stimulation was analysed as stated in Methods. *p<0.05, compared to cells transfected with control

siRNA. http://gut.bmj.com/

pieces of evidence support our identification of AFTPH as a Figure 6 Aftiphilin (AFTPH) was the downstream target of miR-133α downstream target. Incubation of human colonic epi- neurotensin (NT)-induced miR-133α upregulation. (A) Conservative 0 thelial cells with NT decreases AFTPH mRNA levels, as well as miR-133α binding sites were identified in 3 untranslated region (UTR) 0 AFTPH 3 UTR-regulated luciferase activity. Most importantly, region of aftiphilin across different species. (B) Levels of miR-133α in expression of AFTPH with a deleted miR-133α binding on September 29, 2021 by guest. Protected copyright. NCM460-neurotensin receptor 1 (NTR1) cells transfected with 0 as-miR-133α and its control was analysed by qPCR analysis. *p<0.05, sequence on its 3 UTR is no longer downregulated in response compared to cells stimulated by vehicle. (C) AFTPH 30UTR-driven to NT exposure. The presence of AFTPH has not been previ- luciferase activities from NCM460-NTR1 cells transfected with ously recognised in the intestine, and its cellular function is not as-miR-133α and its control were measured after NT stimulation. well characterised. AFTPH is essential to intracellular trafficking *p<0.05, compared to cells stimulated by vehicle. (D) Luciferase activity with binding sites for and adaptor (AP-1, 0 driven by AFTPH 3 UTR with or without miR-133α binding sequence AP-2).52 AFTPH is localised in trans-golgi network (TGN),53 was measured after NT stimulation. *p<0.05, compared to cells 0 and its gene silencing leads to unregulated exocytosis of stimulated by vehicle. (E) AFTPH 3 UTR-driven luciferase activities from Weibel-Palade bodies in endothelial cells54 without altering HEK293 cells transfected with miR-133α precursor (miR-133α) and its TGN morphology.55 However, the role of AFTPH or TGN in control were measured. *p<0.05, compared to cells transfected with fl control miRNA precursor. (F) Immunoblot analysis of AFTPH expression proin ammatory signalling cascades has not been investigated. fi in NCM460-NTR1 cells 30 min and 6 h after NT exposure in the Here, we demonstrate that AFTPH levels are signi cantly down- presence or absence of SR48962 (10 nM). (G) AFTPH levels in colon regulated in colon tissues from patients with UC, as well as in tissues from C57BL6/J mice intracolonically administered with NT and the colon of mice with TNBS-induced and DSS-induced colitis. its control and (H) from C57BL6/J mice intracolonically administered At the cellular level, AFTPH gene silencing activates the NF-κB with as-miR-133α and its control with or without trinitrobenzene pathway and promotes proinflammatory cytokine IL-1β produc- sulfonic acid treatment were analysed using qPCR analysis. *p<0.05, tion in vitro. NF-κB activation is involved in IL-1β expression56 compared to mice administered with control as-miRNA. and administration of NF-κB inhibitor reduce IL-1β production and ameliorates colonic inflammation in mice.57 Thus, dysregu- and in subepithelial cells of the colonic mucosa, participation of lated AFTPH expression may modulate proinflammatory miR-133α in inflammatory signalling in colonic lamina propria responses, representing a novel regulator in proinflammatory cell types cannot be excluded. signalling pathways and colitis development. Moreover, mea- We identified a novel miR-133α target, AFTPH, in which its surements of its expression levels, together with measurements expression level is regulated by NT/NTR1 interactions. Several of the levels of miR-133α, may serve as biomarkers in UC.

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6 Brun P, Mastrotto C, Beggiao E, et al. Neuropeptide neurotensin stimulates intestinal wound healing following chronic intestinal inflammation. Am J Physiol Gut: first published as 10.1136/gutjnl-2014-307329 on 11 August 2014. Downloaded from Gastrointest Liver Physiol 2005;288:G621–9. 7 Zhao D, Keates AC, Kuhnt-Moore S, et al. Signal transduction pathways mediating neurotensin-stimulated interleukin-8 expression in human colonocytes. J Biol Chem 2001;276:44464–71. 8 Sundman L, Saarialho-Kere U, Vendelin J, et al. receptor 1 expression in the intestine and skin--putative role in hormone secretion. Neurogastroenterol Motil 2010;22:79–87, e30. 9 da Silva L, Neves BM, Moura L, et al. Neurotensin downregulates the pro-inflammatory properties of skin dendritic cells and increases epidermal growth factor expression. Biochim Biophys Acta 2011;1813:1863–71. 10 Bakirtzi K, Hatziapostolou M, Karagiannides I, et al. Neurotensin signaling activates microRNAs-21 and -155 and Akt, promotes tumor growth in mice, and is increased in human colon tumors. Gastroenterology 2011;141:1749–61.e1. 11 Margolis KG, Gershon MD. and inflammatory bowel disease. Curr Opin Gastroenterol 2009;25:503–11. 12 Zhao D, Pothoulakis C. Effects of NT on gastrointestinal motility and secretion, and role in intestinal inflammation. 2006;27:2434–44. 13 Bugni JM, Rabadi LA, Jubbal K, et al. The neurotensin receptor-1 promotes tumor development in a sporadic but not an inflammation-associated mouse model of – Figure 8 Levels of miR-133α and aftiphilin (AFTPH) were significantly colon cancer. Int J Cancer 2012;130:1798 805. 14 McKenna LB, Schug J, Vourekas A, et al. MicroRNAs control intestinal epithelial dysregulated in patients with ulcerative colitis (UC). (A) Levels of α differentiation, architecture, and barrier function. Gastroenterology miR-133 and AFTPH in colon tissues from normal and patients with 2010;139:1654–64, 1664 e1. UC were analysed using qPCR analysis. (B) Representative images from 15 Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell immunohistochemistry against AFTPH and villin of colon tissues from 2009;136:215–33. normal and patients with UC. Scale: 50 μm. 16 Wu F, Zikusoka M, Trindade A, et al. MicroRNAs are differentially expressed in ulcerative colitis and alter expression of macrophage inflammatory peptide-2 alpha. – Overall, our results represent a previously unrecognised para- Gastroenterology 2008;135:1624 35 e24. 17 Bian Z, Li L, Cui J, et al. Role of miR-150-targeting c-Myb in colonic epithelial digm whereby activation of a (NTR1) in disruption during dextran sulphate sodium-induced murine experimental colitis and the colonic mucosa stimulates the expression of a miR human ulcerative colitis. J Pathol 2011;225:544–53. (miR-133α) that modulates the development of colitis. The 18 Fasseu M, Treton X, Guichard C, et al. Identification of restricted subsets of mature therapeutic potential of miR-133α silencing and stabilisation of microRNA abnormally expressed in inactive colonic mucosa of patients with fl colonic AFTPH levels against colitis deserves further in ammatory bowel disease. PLoS ONE 2010;5:pii:e13160. 19 Takagi T, Naito Y, Mizushima K, et al. Increased expression of microRNA in the investigation. inflamed colonic mucosa of patients with active ulcerative colitis. J Gastroenterol and Hepatol 2010;25:S129–33. Contributors Overall project design and hypotheses were developed by IKML and 20 Wu F, Zhang S, Dassopoulos T, et al. Identification of microRNAs associated with CP. IKML conducted experiments, analysed the data and wrote the manuscript. KB ileal and colonic Crohn’s disease. Inflamm Bowel Dis 2010;16:1729–38. http://gut.bmj.com/ generated the human colon epithelial cells overexpressing NTR1, NCM460-NTR1. DH 21 Josse C, Bouznad N, Geurts P, et al. Identification of a microRNA and AO collected and provided sera from UC and control patients and for analysing landscape targeting the PI3K/Akt signaling pathway in inflammation-induced these new results now included in the revised manuscript. CP, DI and CP colorectal carcinogenesis. Am J Physiol Gastrointest Liver Physiol 2014;306: contributed to experimental design and provided animals and reagents. All authors G229–43. fi participated in revising the manuscript and agreed to the nal version. CP and DI 22 Wang H, An H, Wang B, et al. miR-133a represses tumour growth and metastasis supervised the overall project. in colorectal cancer by targeting LIM and SH3 protein 1 and inhibiting the MAPK Funding This work was supported by NIH grant DK60729 (CP), the pathway. Eur J Cancer 2013;49:3924–35. Neuroendocrine Assay Core, NIDDK P50 DK 64539 (CP), the Blinder Research 23 Arndt GM, Dossey L, Cullen LM, et al. Characterization of global microRNA Foundation for Crohn’s Disease (IKML), and the Crohn’s and Colitis Foundation of expression reveals oncogenic potential of miR-145 in metastatic colorectal cancer. on September 29, 2021 by guest. Protected copyright. America (KB). BMC Cancer 2009;9:374. 24 Sarver AL, French AJ, Borralho PM, et al. Human colon cancer profiles show Competing interests None. differential microRNA expression depending on mismatch repair status and are Provenance and peer review Not commissioned; externally peer reviewed. characteristic of undifferentiated proliferative states. BMC Cancer 2009;9:401. 25 Dong Y, Zhao J, Wu CW, et al. Tumor suppressor functions of miR-133a in Open Access This is an Open Access article distributed in accordance with the colorectal cancer. Mol Cancer Res 2013;11:1051–60. 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